Chitosanases are enzymes hydrolysing chitosan, a β-1,4 linked D-glucosamine bio-polymer. Chitosan oligosaccharides have numerous emerging applications and chitosanases can be used for industrial enzymatic hydrolysis of chitosan. These extracellular enzymes, produced by many organisms including fungi and bacteria, are well studied at the biochemical and enzymatic level but very few works were dedicated to the regulation of their gene expression.
Chitosan, a partly N-deacetylated form of chitin, is naturally found in the cell walls of fungi, especially in Zygomycetes (Mucor sp., Rhizopus sp.), and in the green algae Chlorophyceae (Chlorella sp.). Chitosan, is a polysaccharide made of β-1,4-linked D-glucosamine (GlcN) units with a variable content of N-acetyl-D-glucosamine (GlcNAc) units. Chitosan is produced at industrial scale by alkaline deacetylation of chitin, originating mainly from crustacean shells. This polysaccharide, almost unique among natural polymers for its amino groups that remain positively charged in mild acidic solutions, is the subject of numerous works oriented towards its numerous emerging applications in medicine, agriculture, dietetics, environment protection and several other fields. Chitosan is also a valuable source of GlcN, a neutraceutical used as a therapeutic agent in osteoarthritis. Many properties of chitosan, especially in biological applications are dependent on its molecular weight, i.e. on its degree of polymerization.
The very short derivatives of chitosan—the chito-oligosaccharides are of particular interest, due to their increased solubility in aqueous solutions and their specific biological activities. To obtain chitosan chain of varying degrees of polymerization, several chemical and physical techniques were investigated. Enzymatic techniques with either free or immobilized chitinase or chitosanase enzymes are also intensively studied. Chitosanase production has been found in many microorganisms, bacteria or fungi. The enzymes so far characterized at the primary sequence level belong to seven families of glycoside hydrolases: GH3, GH5, GH7, GH8, GH46, GH75 and GH80. While these enzymes are endo-hydrolases, their mechanism could potentially be transformed into exo-type by protein engineering as shown for the GH46 chitosanase from Bacillus circulars MH-K1. Chitosan can be also hydrolyzed by enzymes acting by an exo-mechanism generating GlcN monomers. The chitosanases from Streptomyces have been widely studied in various aspects of structure-function relationships. Usually, these chitosanases are produced in the heterologous host Streptomyces lividans via the multi-copy vector pFD666. However, very few works have been dedicated to the regulation of chitosanase gene expression in the native and/or heterologous hosts. Most studies were limited to the follow up of chitosanase production in various culture media. An efficient production of CsnN106 or CsnN174 chitosanases in Streptomyces lividans TK24 is strictly dependent on the addition of chitosan or its derivatives to the culture medium indicating that these foreign genes are still subjected to some kind of chitosan-dependent regulation in the heterologous host. However, the addition of chitosan as a component in any culture medium is not without problems due to the well known anti-microbial properties of this polysaccharide which can slow down the bacterial growth.
Microbiological studies and the analysis of sequenced genomes showed that chitosanases are widespread among filamentous fungi and Gram-positive bacteria, particularly in bacilli and actinobacteria. In Streptomyces, well-studied chitosanases belong to glycoside hydrolase families GH2, GH5, GH46, and GH75. Putative chitosanases from these families, as well as from GH8 (characterized mainly from Gram-positive bacili) are found in many recently sequenced actinomycete genomes (CaZy database). Streptomyces lividans is an actinomycete isolated from soil, commonly used as heterologous host for production of proteins in an extracellular mode, including the well-studied chitosanase from Streptomyces sp. N174 (CsnN174). Until the publication of the genome sequence of S. coelicolor A3(2) and, more recently, of the S. lividans genomic contigs (GenBank accession no. ACEY010000), these two closely related species were thought to be devoid of chitosanase activity because they grew very poorly on media with chitosan and no chitosanase activity was detected in their cultures. However, genes encoding putative chitosanases of the GH46 family are present in both genomes: SCO0677 (csnA) and SCO2024 (csnB) in Streptomyces coelicolor A3(2) and the almost identical genes SSPG—06922 (genomic coordinate 7.62 Mb) and SSPG—05520 (genomic coordinate 6.14 Mb) in S. lividans TK24. The biochemical properties of CsnA from S. coelicolor A3(2) have been studied in detail recently. In vivo studies performed with S. lividans TK24 have shown that CsnA is produced at a very low level (in the range of milliunits per ml), explaining the lack of chitosanase detection by earlier, less-sensitive techniques. Despite this low expression level, the deletion of csnA resulted in increased sensitivity to the antimicrobial effect of chitosan. While there are numerous reports on biochemical properties of chitosanases, knowledge about the regulation of chitosanase gene expression is very scarce. In contrast, the genetic regulation of the degradation of chitin, the N-acetylated form of chitosan, has been extensively studied in Streptomyces. Members of this genus play an important part in chitin degradation in soil and produce a wide array of chitinases and chitin-binding proteins. The regulation of chitinase (chi) gene expression in Streptomyces is rather complex, and as many as four different mechanisms have been identified, some of them linked to more general phenomena such as carbon catabolite repression, antibiotic production, and morphogenesis through the chitin-derived monomer N-acetyl-D-glucosamine (GlcNAc). The Cpb1 regulator controls the expression of the chiA gene in S. lividans. The two-component system ChiS/ChiR participates to the genetic regulation of chiC gene of S. coelicolor. Reg1, the negative regulator of α-amylase genes in S. lividans, seems also to be involved in the genetic regulation of chitinase genes. Finally DasR, a member of the HutC/GntR subfamily, regulates the expression of some chitinase genes through interaction with the dre motif in S. coelicolor. DasR also has a more global effect on other genes involved in GlcNAc metabolism.
It would be highly desirable to be provided with an expression system for a chitosanase which is not dependant on the presence of chitosan in the culture medium. It would be desirable to be provided with an expression system which would allow for the expression of endogenous as well as exogenous chitosanase. It would also be highly desirable to be provided with an expression system for a chitosanase which limits or avoids the production of protease in the culture medium. It would further be desirable, for pharmaceutical applications, to be provided with an expression system for a chitosanase which can be cultured in a defined medium.